Disease-modifying effects of metabolic perturbations in ALS/FTLD

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two fatal neurodegenerative disorders with considerable clinical, pathological and genetic overlap. Both disorders are characterized by the accumulation of pathological protein aggregates that contain a number of proteins, most notably TAR DNA binding protein 43?kDa (TDP-43). Surprisingly, recent clinical studies suggest that dyslipidemia, high body mass index, and type 2 diabetes mellitus are associated with better clinical outcomes in ALS. Moreover, ALS and FTLD patients have a significantly lower incidence of cardiovascular disease, supporting the idea that an unfavorable metabolic profile may be beneficial in ALS and FTLD. The two most widely studied ALS/FTLD models, super-oxide dismutase 1 (SOD1) and TAR DNA binding protein of 43 kDA (TDP-43), reveal metabolic dysfunction and a positive effect of metabolic strategies on disease onset and/or progression. In addition, molecular studies reveal a role for ALS/FTLD-associated proteins in the regulation of cellular and whole-body metabolism. Here, we systematically evaluate these observations and discuss how changes in cellular glucose/lipid metabolism may result in abnormal protein aggregations in ALS and FTLD, which may have important implications for new treatment strategies for ALS/FTLD and possibly other neurodegenerative conditions.     

The minor spliceosome could be the major key for FUS/TLS mutants in ALS

Despite its name, minor spliceosome alterations are often involved in human disease origin. Work by Reber et al ( 2016 ) in this issue of The EMBO Journal now demonstrates a connection between minor spliceosome components and FUS/TLS, one of the major proteins aggregating in the brain of patients affected by amyotrophic lateral sclerosis (ALS). This finding has important implications as it extends the spectrum of diseases where minor spliceosome plays a role. It may also represent a new opportunity for specific therapeutic targets.     

Understanding the role of TDP-43 and FUS/TLS in ALS and beyond

Dominant mutations in two DNA/RNA binding proteins, TDP-43 and FUS/TLS, are causes of inherited Amyotrophic Lateral Sclerosis (ALS). TDP-43 and FUS/TLS have striking structural and functional similarities, implicating alterations in RNA processing as central in ALS. TDP-43 has binding sites within a third of all mouse and human mRNAs in brain and this binding influences the levels and splicing patterns of at least 20% of those mRNAs. Disease modeling in rodents of the first known cause of inherited ALS-mutation in the ubiquitously expressed superoxide dismutase (SOD1)-has yielded non-cell autonomous fatal motor neuron disease caused by one or more toxic properties acquired by the mutant proteins. In contrast, initial disease modeling for TDP-43 and FUS/TLS has produced highly varied phenotypes. It remains unsettled whether TDP-43 and FUS/TLS mutants provoke disease from a loss of function or gain of toxicity or both. TDP-43 or FUS/TLS misaccumulation seems central not just to ALS (where it is found in almost all instances of disease), but more broadly in neurodegenerative disease, including frontal temporal lobular dementia (FTLD-U) and many examples of Alzheimer's or Huntington's disease.     

Modeling ALS and FTLD proteinopathies in yeast: An efficient approach for studying protein aggregation and toxicity

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.     

Modeling ALS and FTLD proteinopathies in yeast: An efficient approach for studying protein aggregation and toxicity

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.Key words: amyotrophic lateral sclerosis, ALS, frontotemporal lobar degeneration, FTLD, yeast, amyloid, prion, FUS, TDP-43, SOD1     

Scintillation proximity assay of arginine methylation

Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein posttranslational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, (3)H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.     

Critical role for arginine methylation in adenovirus-infected cells

During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.     

The cardiac sodium channel is post-translationally modified by arginine methylation

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.     

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.     

Deciphering arginine methylation: Tudor tells the tale

Proteins can be modified by post-translational modifications such as phosphorylation, methylation, acetylation and ubiquitylation, creating binding sites for specific protein domains. Methylation has pivotal roles in the formation of complexes that are involved in cellular regulation, including in the generation of small RNAs. Arginine methylation was discovered half a century ago, but the ability of methylarginine sites to serve as binding motifs for members of the Tudor protein family, and the functional significance of the protein-protein interactions that are mediated by Tudor domains, has only recently been appreciated. Tudor proteins are now known to be present in PIWI complexes, where they are thought to interact with methylated PIWI proteins and regulate the PIWI-interacting RNA (piRNA) pathway in the germ line.     

The story of protein arginine methylation: characterization,regulation, and function

Introduction: Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery.

Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases.

Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates.